A modular spring-loaded actuator for mechanical activation of membrane proteins.

How cells respond to mechanical forces by converting them into biological signals underlie crucial cellular processes. Our understanding of mechanotransduction has been hindered by technical barriers, including limitations in our ability to effectively apply low range piconewton forces to specific mechanoreceptors on cell membranes without laborious and repetitive trials. To overcome these challenges we introduce the Nano-winch, a robust, easily assembled, programmable DNA origami-based molecular actuator. The Nano-winch is designed to manipulate multiple mechanoreceptors in parallel by exerting fine-tuned, low- piconewton forces in autonomous and remotely activated modes via adjustable single- and double-stranded DNA linkages, respectively. Nano-winches in autonomous mode can land and operate on the cell surface. Targeting the device to integrin stimulated detectable downstream phosphorylation of focal adhesion kinase, an indication that Nano-winches can be applied to study cellular mechanical processes. Remote activation mode allowed finer extension control and greater force exertion. We united remotely activated Nano-winches with single-channel bilayer experiments to directly observe the opening of a channel by mechanical force in the force responsive gated channel protein, BtuB. This customizable origami provides an instrument-free approach that can be applied to control and explore a diversity of mechanotransduction circuits on living cells.

Palmitoyl-carnitine increases RyR2 oxidation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes: Role of adenine nucleotide translocase.

Long chain fatty acids bind to carnitine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulation is associated with severe cardiac arrhythmia including ventricular tachycardia or fibrillation. We thus hypothesized that palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca(2+) dependent-arrhythmia. In isolated cardiac mitochondria from C57Bl/6 mice, application of 10µM PC decreased adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial reactive oxygen species (ROS) production, measured with MitoSOX Red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongkrekic acid (20 µM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation, S-nitrosylation and dissociation of FKBP12.6 from RyR2, and therefore increased sarcoplasmic reticulum (SR) Ca(2+) leak. ANT inhibition or anti-oxidant strategy (N-acetylcysteine) prevented SR Ca(2+) leak, FKBP12.6 depletion and RyR2 oxidation/S-nitrosylation induced by PC. Finally, both bongkrekic acid and NAC significantly reduced spontaneous Ca(2+) wave occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca(2+) handling in a ROS-dependent pathway, demonstrating a new pathway whereby altered FA metabolism may contribute to the development of ventricular arrhythmia in pathophysiological conditions.

A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a structural modification of the major porin.

In Enterobacter aerogenes, multidrug resistance involves a decrease in outer membrane permeability associated with changes in an as yet uncharacterized porin. We purified the major porin from the wild-type strain and a resistant strain. We characterized this porin, which was found to be an OmpC/OmpF-like protein and analysed its pore-forming properties in lipid bilayers. The porin from the resistant strain was compared with the wild-type protein and we observed (i) that its single-channel conductance was 70% lower than that of the wild type; (ii) that it was three times more selective for cations; (iii) a lack of voltage sensitivity. These results indicate that the clinical strain is able to synthesize a modified porin that decreases the permeability of the outer membrane. Mass spectrometry experiments identified a G to D mutation in the putative loop 3 of the porin. Given the known importance of this loop in determining the pore properties of porins, we suggest that this mutation is responsible for the novel resistance mechanism developed by this clinical strain, with changes in porin channel function acting as a new bacterial strategy for controlling beta-lactam diffusion via porins.

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli.

The proteolytic fragments of OprFs of Pseudomonas aeruginosa and Pseudomonas fluorescens were identified, respectively, as the first 175 and 177 amino acids from the N-terminal domain. They induced ion channels after reincorporation into planar lipid bilayers (85 and 75 pS, respectively, in 1 M NaCl). A similar conductance value (72 pS) was found for the eight beta-strand OmpA N-terminal domain (OmpA171) of Escherichia coli. We conclude that the N-terminal domain of OprFs is sufficient to induce ion channels and the comparison with OmpA171, provides strong evidence of the existence of an eight-stranded beta-barrel in the N-terminal domain of OprFs.

Channel formation by FhaC, the outer membrane protein involved in the secretion of the Bordetella pertussis filamentous hemagglutinin.

Many virulence factors of pathogenic microorganisms are presented at the cell surface. However, protein secretion across the outer membrane of Gram-negative bacteria remains poorly understood. Here we used the extremely efficient secretion of the Bordetella pertussis filamentous hemagglutinin (FHA) to decipher this process. FHA secretion requires a single specific accessory protein, FhaC, the prototype of a family of proteins necessary for the extracellular localization of various virulence proteins in Gram-negative bacteria. We show that FhaC is heat-modifiable and localized in the outer membrane. Circular dichroism spectra indicated that FhaC is rich in beta-strands, in agreement with structural predictions for this protein. We further demonstrated that FhaC forms pores in artificial membranes, as evidenced by single-channel conductance measurements through planar lipid bilayers, as well as by liposome swelling assays and patch-clamp experiments using proteoliposomes. Single-channel conductance appeared to fluctuate very fast, suggesting that the FhaC channels frequently assume a closed conformation. We thus propose that FhaC forms a specific beta-barrel channel in the outer membrane for the outward translocation of FHA.

A hexameric transmembrane pore revealed by two-dimensional crystallization of the large mechanosensitive ion channel (MscL) of Escherichia coli.

We have established a reconstitution method of the detergent-solubilized recombinant large mechanosensitive ion channel of Escherichia coli (MscL) that yielded two-dimensional crystals. For that purpose, we have developed a new protocol using Triton X-100 to solubilize and purify the MscL protein. This protocol not only allowed an increase in the protein yield but also made it possible to obtain a homogeneous delipidated and reproducible preparation of the purified protein. When examined by the patch-clamp method MscL channels were found to be fully functional, exhibiting characteristic conductance and activation by pressure. For electron crystallography the homogeneous Triton X-100-purified recombinant MscL was further reconstituted at low lipid-to-protein ratios using Bio-Beads SM2 to remove the detergent. Two-dimensional crystals, exhibiting a p6 plane group symmetry, have been produced and examined by negative stain electron microscopy. Image processing of selected micrographs yielded a projection map at 15-A resolution that provided the first explicit structural information about the molecular boundary and homohexameric organization of the MscL channels in the membrane bilayer.

Mechanosensitive ion channels of the archaeon Haloferax volcanii.

Mechanosensitive (MS) ion channels have been documented in a variety of cells belonging to Eukarya and Eubacteria. We report the novel finding of two types of MS ion channels in the cell membrane of the halophilic archaeon Haloferax volcanii, a member of the Archaea that comprise the third phylogenetic domain. The two channels, MscA1 and MscA2, differed in their kinetic properties with MscA1 exhibiting more frequent open-closed transitions than MscA2. Both channels have large conductances that rectify between -40 mV and +40 mV where the conductance of MscA1 ranged from 380 to 680 picosiemens, whereas MscA2 ranged from 850 to 490 picosiemens. Both channels were blocked by submillimolar gadolinium. In addition, the channels of either membrane vesicles or detergent-solubilized membrane proteins remained functional upon reconstitution into artificial liposomes, a result that indicates that these channels are activated by mechanical force transmitted via the lipid bilayer alone. Subsequently a 37-kDa protein corresponding to the MscA1 channel activity was purified. With the possible functional similarity to bacterial MS channels, our finding of MS channels in Archaea emphasizes the ubiquity and importance of these channels in all domains of the evolutionary tree.

Role of the constriction loop in the gating of outer membrane porin PhoE of Escherichia coli.

Porins form voltage-gated channels in the bacterial outer membrane. These proteins are composed of three identical subunits, each forming a 16-stranded beta-barrel. In this study, the role in voltage gating of a loop that forms a constriction within the pore was studied. The channel characteristics of mutant PhoE porins, in which the tip of the constriction loop was connected to the barrel wall, were determined. Whereas the properties of several mutant channels were changed, all of these channels could still be closed at high potential, showing that a gross movement of the constriction loop within the channel is not implicated in voltage gating.

Voltage sensing in the PhoE and OmpF outer membrane porins of Escherichia coli: role of charged residues.

The porins PhoE and OmpF form anion and cation-selective pores, respectively, in the outer membrane of Escherichia coli. Each monomer of these trimeric proteins consists of a 16-stranded beta-barrel, which contains a constriction at half the height of the channel. The functional significance of a transverse electrical field that is formed by charged amino acid residues within the constriction zone was investigated. For this purpose, the PhoE residues R37, R75, K18 and E110 were substituted by neutral amino acids. The mutant pores allowed an increased permeation of beta-lactam antibiotics across the outer membrane in vivo, although the single channel conductance, measured in planar lipid bilayers, was not increased or even slightly decreased. Replacement of the positively charged residues resulted in a decreased voltage sensitivity, whereas the substitution of a negatively charged residue resulted in an increased voltage sensitivity. Similar substitutions in OmpF caused the opposite effects, i.e. the substitution of positive and negative charges resulted in increased and decreased voltage sensitivity, respectively. Together, the results suggest that opposite charges, i.e. positive charges in anion-selective and negative charges in cation-selective porins, act as sensors for voltage gating.

Pore functioning of outer membrane protein PhoE of Escherichia coli: mutagenesis of the constriction loop L3.

Each monomer of the trimeric outer membrane porin PhoE of Escherichia coli consists of a 16-stranded beta-barrel with short turns at the periplasmic side and large loops at the cell surface. One of these loops, L3, is folded inside the beta-barrel and forms a constriction within the channel. Therefore, it is assumed to play an important role in the permeability properties of this general diffusion pore. Several site-directed mutations were introduced in loop L3 to investigate its function. The loop L3 contains a short alpha-helix and, at the tip of the loop, a highly conserved PEFGG sequence. The alpha-helix was deleted and the two glycines in the PEFGG sequence were either replaced by alanines or deleted. A serine residue, supposed to play an indirect role in the anion selectivity of the pore, was removed. The mutant porins were analysed both in vitro and in vivo. The results suggest that flexibility of the third loop is important for solute passage and that this flexibility is determined by the two glycine residues in the PEFGG sequence. Furthermore, the alpha-helix is probably important for the folding of the protein. The supposed involvement of Ser115 (Ser121A in OmpF nomenclature) in anion selectivity was confirmed.

Structural and functional characterization of OmpF porin mutants selected for larger pore size. II. Functional characterization.

The effects on the channel characteristics of four single amino acid substitutions in OmpF porin and of a deletion mutant in the constriction loop L3 have been studied. These mutations are all located in the narrow section of the channel of the protein that forms pores across the outer membrane of Escherichia coli. The single channel conductance of the deletion mutant (Delta109-114) is decreased by one third, whereas the point mutations do not exhibit significant deviations from that of the wild-type protein. The mutants exhibit drastic changes in ion selectivities. In the wild-type protein, the critical threshold potential (Vc), above which channels close reversibly, exhibits a strong pH dependence, with a titration point of approximately pH 7.7, which is abolished in all mutants studied here. Diffusion of six monosaccharides is little affected in the point mutants, while four disaccharides are taken up at highly increased rates by the deletion mutant. The functional results, presented here, are correlated to the x-ray structures of the mutants (Lou, K.-L., Saint, N., Prilipov, A., Rummel, G., Benson, S.A., Rosenbusch, J.P., and Schirmer, T. (1996) J. Biol. Chem. 271, 20669-20675). In most, but not all, cases, the structural changes explain the functional alterations observed.

Structural and functional characterization of OmpF porin mutants selected for larger pore size. I. Crystallographic analysis.

OmpF porin is a nonspecific pore protein from the outer membrane of Escherichia coli. Previously, a set of mutants was selected that allow the passage of long maltodextrins that do not translocate through the wild-type pore. Here, we describe the crystal structures of four point mutants and one deletion mutant from this set; their functional characterization is reported in the accompanying paper (Saint, N., Lou, K.-L., Widmer, C., Luckey, M., Schirmer, T., Rosenbusch, J. P. (1996) J. Biol. Chem. 271, 20676-20680). All mutations have a local effect on the structure of the pore constriction and result in a larger pore cross-section. Substitution of each of the three closely packed arginine residues at the pore constriction (Arg-42, Arg-82, and Arg-132) by shorter uncharged residues causes rearrangement of the adjacent basic residues. This demonstrates mutual stabilization of these residues in the wild-type porin. Deletion of six residues from the internal loop (Delta109-114) results in disorder of seven adjacent residues but does not alter the structure of the beta-barrel framework. Thus, the large hollow beta-barrel motif can be regarded as an autonomous structure.

Characterization and ion channel activities of novel antibacterial proteins from the skin mucosa of carp (Cyprinus carpio).

A detergent-solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels after reconstitution into planar lipid bilayers. A differential extraction using a non-ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31-kDa and 27-kDa proteins. In contrast to the 27-kDa protein, which was glycosylated, the 31-kDa did not bind to concanavalin A. The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus. The main unit conductance levels were about 900 pS for the 27-kDa protein and 500 pS for the 31-kDa protein, and selectivity measurements gave Pcl/Pk ratios of 0.6 and 1.0, respectively. These proteins had large potent microbicidal activities (0.018-0.18 microM) against different strains of gram-negative and gram-positive bacteria. This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane. To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia; Micrococcus luteus; Micrococcus roseus; Flavobacterium sp.; Aeromonas hydrophila. Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities. Furthermore, microsequencing analysis showed that the 31-kDa protein was protected on its N-terminal extremity in contrast to the 27-kDa protein, which had a 19-amino-acid sequence. This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins. These results suggest that these novel proteins could be involved in antibacterial defense processes in fish.

Replacement of the sole histidinyl residue in OmpF porin from E. coli by threonine (H21T) does not affect channel structure and function.

The sole histidine residue in OmpF porin was replaced by threonine using site-directed mutagenesis. This exchange affected neither channel properties nor channel structure, as determined by X-ray analysis to 3.2 A. Conductance and critical voltage (Vc) were observed in the pH range 4.3-9.4, with results indistinguishable from those observed in the wild-type protein. The validity of these observations is supported by the independence of the methods used, and by the fact that mutants in residues located in the channel constriction yielded significantly different values from wild-type protein. The binding of a glycolipid molecule might be affected.

Channel-forming properties and structural homology of major outer membrane proteins from Pseudomonas fluorescens MFO and OE 28.3.

The major outer membrane proteins (OprF) from Pseudomonas fluorescens MFO and OE 28.3 were purified by a new method involving native electrophoresis in octyl-polyoxyethylene media. Both proteins, characterized by the same size, heat-modifiability and N-terminal sequence were re-incorporated in virtually solvent-free planar lipid bilayers. They displayed very similar channel-forming properties: the major conductance level was between 250 pS and 270 pS in 1 M NaCl. From experiments of zero-current potential, both porins were determined weakly cation selective. Amplification by PCR and sequencing of the oprF gene of strain MFO allowed to point out 94% identity between the amino acid sequences of these two OprFs isolated from ecological niches as different as milk (strain MFO) and soil (strain OE 28.3).

Ionophore properties of OmpA of Escherichia coli.

Both porins OmpA1, from wild-strain K12 Escherichia coli, and OmpA2, from a K12 derivative deficient in both OmpF and OmpC, are able to form ion channels in virtually solvent-free membranes. The conductance has been shown to vary in a discrete fashion with different single increment values especially with OmpA2. This behaviour seems to indicate, beside monomers, the presence of aggregates of different sizes. The estimated small pore diameter (0.6-0.7 nm) for the monomeric would explain the weak permeability of this narrow channel toward different solutes. OmpA protein, from experiments of ion selectivity and zero-current potential, is determined weakly anion selective.